1. Reading Alberts Chapter 8 p. 376-385 Alberts Chapter 12 p. 561-567
Lodish Chapter 11 p RNA transport
Reading
Alberts Chapter 8 p
Alberts Chapter 12 p
Lodish Chapter 11 p RNA transport

2. Cooper 6-32
Activation
Acetylation of histones correlates with actively transcribed genes
Acetylation reduces the net positive charge of the histones
HMG14 or 17 compete with H1 histones for binding to the nucleosome
De-acetylation is involved in turning transcription off
Acetylation of core histones decreases affinity for DNA by reducing the overall positive charge of the histones. Deacetylation restores normal order.
Activation
Acetylation of histones correlates with actively transcribed genes
Acetylation reduces the net positive charge of the histones
HMG14 or 17 (high mobility group) compete with H1 histones for binding to the nucleosome
De-acetylation is involved in turning transcription off

3. Chromosome organization

4. Metaphase chromosome Chromatids Telomeres Centromere Alberts 18-15
Holds sister chromatids together
Attachment of kinetochore during mitosis
Metaphase chromosome
Chromatids
Telomeres
Centromere
Holds sister chromatids together
Attachment of kinetochore during mitosis
Alberts 18-15

5. Alberts 8-4 Origins of replication-sites where DNA replication begins.
Telomeres are specialized sequences at the end of linear chromosomes that ensure that genetic material is not lost during replication.
Centromeres hold chromatids together prior to cell division and associate with kinetochores.
Origins of replication-sites where DNA replication begins.
Telomeres are specialized sequences at the end of linear chromosomes that ensure that genetic material is not lost during replication.
Centromeres hold chromatids together prior to cell division and associate with kinetochores.
Why hold the sister chromatids together?
May be mechanism that ensures the chromosome is only copied one.
What might happen if you have two centromeres?
Centromere in combination with the kinetochore ensures that each daughter cell receives one copy of the genetic information. Two centromeres could cause the chromosome to split in half the wrong way.

6. Kinetochore Attachment site for spindle microtubules
DNA/ protein structure
Required for proper chromosome segregation
Kinetochore
Attachment site for spindle microtubules
DNA/ protein structure
Required for proper chromosome segregation
Alberts 18-16

7. Yeast centromere has been characterized.
Alberts 18-17
Yeast centromere has been characterized.
Simple DNA sequence that binds to microtuble
Yeast centromere has been characterized.
Simple DNA sequence that binds to microtuble

8. Distinctive banding pattern for each chromosome
Chromosome Banding
Chromomeres
Distinctive banding pattern for each chromosome
Hoechst (G bands--AT rich sequence)
Olivomycin (R bands--GC rich sequence)
Giemsa
Feulgen reagent
Chromosome Banding
Chromomeres--bead-like, localized thickenings of chromosomes
reproducible localization
nature unknown
Distinctive banding pattern for each chromosome indicative of spatial organization.
Hoechst (G bands--AT rich sequence)
Olivomycin (R bands--GC rich sequence)
Giemsa
Feulgen reagent
Alberts 8-31

9. Domains of replication
Metabolically label cells with bromodeoxyuridine
Substitutes for thymidine
Alters staining of G bands or can use antibodies to detect
Synthesis occurs in distinct domains during S phase
Domains of replication
Metabolically label cells with bromodeoxyuridine
Substitutes for thymidine
Alters staining of G bands or can use antibodies to detect
Synthesis occurs in distinct domains during S phase
Alberts 8-37

10. Examine position of a gene on a chromosome
In situ hybridization
Examine position of a gene on a chromosome
Examine position of chromosome in nucleus
Examine distribution of transcript in cytoplasm
Basic hybridization technique
Label probe
Hybridize via base pairing to target
In situ hybridization
Examine position of a gene on a chromosome
gene specific probe
Examine position of chromosome in nucleus
Fragmented chromosomal DNA or gene specific probe known to be on chromosome of interest
Examine distribution of transcript in cytoplasm
cDNA specific probe
Basic hybridization technique
Label probe
Fluorescence, radioactive, gold, distinctive Ag
Hybridize via base pairing to target
Stringency is dictated by buffers, formamide and temperature
Alberts 7-17

11. Result of in situ hybridization
Gene specific probes
Unique labels for each gene
Metaphase chromosome 5
Duplicate spots for each probe
Positions on each pair similar
Result of in situ hybridization
Gene specific probes
Unique labels for each gene
Metaphase chromosome 5
Duplicate spots for each probe
Positions on each pair similar
Alberts 7-19

12. How do we use these techniques to study the interphase chromosomes?
Morphological studies suggest that the DNA is dispersed randomly throughout the nucleoplasm.
How is the DNA organized within the nucleus?
How do we use these techniques to study the interphase chromosomes?
Morphological studies suggest that the DNA is dispersed randomly throughout the nucleoplasm.
How is the DNA organized within the nucleus?
Cooper 8-15

13. Chromatin is organized in a discrete manner in the interphase nucleus
Rabl 1885 suggested that each chromosome occupies a distinct space or territory.
Centromeres and telomeres attached at opposite sides of the nuclear envelope based on orientation during mitosis
Cooper 8-16
Chromatin is organized in a discrete manner in the interphase nucleus
Rabl 1885 suggested that each chromosome occupies a distinct space or territory.
Centromeres and telomeres attached at opposite sides of the nuclear envelope based on orientation during mitosis
Alberts 8-68

14. Rabl observations were confirmed 100 years later by studies of Drosophila embryos
Chromosomal order at the end of mitosis
Indication that interphase chromatin is not entangled mass.
Ordered structure.
Rabl observations were confirmed 100 years later by studies of Drosophila embryos
Chromosomal order at the end of mitosis due to orientation brought about by attachment of the spindles to the kinetochores.
Indication that interphase chromatin is not entangled mass.
Ordered structure.
Alberts 8-68

15. Chromosomes localize to specific domains or territories.
Cooper 8-18
In situ hybridization can be used to analyze the position of chromosomes by using chromosome specific probes. Fluorochrome labeled probes that recognize specific chromosomes are hybridized with interphase cells. The Net result is that each chromosome pair lies within a specific domain within the nucleus.
In situ hybridization performed on each chromosome indicate that each chromosome lies in a discrete domain within the nucleus. Spaces between the chromosomes are believed to be channels for nuclear transport and RNA processing.
Chromosomes localize to specific domains or territories.
Space between the chromosomes may serve as a channel for transport.

16. Nucleolus

17. Nucleolus Fibrillar center contains inactive DNA
Dense fibrillar component contains pre-rRNA synthetic sites
Granular components are sites of of ribosomal subunit assembly
Nucleolus
Fibrillar center contains inactive DNA
Dense fibrillar component contains pre-rRNA synthetic sites
Granular components are sites of of ribosomal subunit assembly
Alberts 8-65

18. The genes encoding the rRNAs are located on 5 different chromosomes
rRNA genes are tandem repeats encoding a 45S precursor RNA
Nucleolar organizing centers.
Contain ribosomal genes
The genes encoding the rRNAs are located on 5 different chromosomes
rRNA genes are tandem repeats of a 45S precursor RNA
Nucleolar organizing centers.
Contain ribosomal genes
The “decondensed” genes encoding the rRNAs are located in the nucleolus of the the interphase nucleus
Alberts 8-63

19. Alberts 8-61
rRNA are expressed from a tandem array of the gene encoding the rRNA precursor. Active transcription of these tandem arrays is displayed in the EM shown above.
Tandem array of rRNA genes results in the amplification of gene expression

20. Proteins of the nucleolus
RNA polymerase I
Nucleolin
RNA binding motifs
Highly phosphorylated
B23
NO38/numatrin/nucleo-phosmin
Fibrillarin
Associates with snoRNPS
snoRNP
Small nucleolar RNPs
over 150 different ones
RNA chaperones, ribonucleases, and guides for rRNA modifications
RNA polymerase I
Nucleolin
RNA binding motifs
Highly phosphorylated
B23
NO38/numatrin/nucleophosmin
Fibrillarin
Associates with snoRNPs
snoRNP
Small nucleolar RNPs
over 150 different ones
RNA chaperones, ribonucleases, and guides for rRNA modifications

21. Cooper 8-28
Ribosomal proteins are synthesized in the cytoplasm and transported back into the nucleus
Ribosomal proteins are synthesized in the cytoplasm and transported back into the nucleus.
Proteins associate with pre-rRNA (45S precursor) prior to modifications and splicing.
Ribosomal subunits are assembled in the nucleolus and transported out of the nucleus through the nuclear pores.

22. The nucleolus is a dynamic structure.
Alberts 8-67
The nucleolus is a dynamic structure.
Changes shape in a cell cycle dependent manner.
The nucleolus is a dynamic structure.
Changes shape in a cell cycle dependent manner.
Three different cells displaying different morphologies of the nucleolus.
Nucleolus is visible by light microscopy.

23. Cell cycle and nucleolar structure
Completely disappears during mitosis
Components associate with chromosomes during mitosis
Self assembles in daughter cells
Cell cycle and nucleolar structure
Completely disappears during mitosis
Components associate with chromosomes during mitosis
Self assembles in daughter cells.
Differences in size reflect differences in the amount of granular component and thus differences in the amount of rRNA synthesis.
Alberts 8-66

24. Nuclear pores Alberts 8-72
Electron micrograph of nuclear pores on the cytoplasmic surface of the nuclear envelope. In some cells the pores are arranged in periodic or order pattern on the nuclear envelope. The pores are a complex structure composed of over 100 different proteins called nucleoporins organized into the nuclear pore complex.
Alberts 8-72

25. Everything that enters or exists the nucleus must pass through the nuclear pores
Passive diffusion
Small proteins (less than 50 kD
Active transport
Utilizes energy
Everything that enters or exists the nucleus must pass through the nuclear pores
Passive diffusion
Active transport
Cooper 8-5

26. Isolated nuclear pore complex
Cooper 8-6
Isolated nuclear pore complex
Mass of 125 million Daltons
Diameter of 120 nm with a central channel
Octagonal arrangement
Electron micrograph of nuclear pores
Extremely large complex --mass is 30 times that of the ribosome
The central channel can open to more than 25 nm in diameter.
Note the octagonal arrangement

27. Scanning electron micrographs of the nuclear pore complex
Lodish 11-28
Scanning electron micrographs of the nuclear pore complex
Cytoplasmic face
Nucleoplasmic face
Nucleoplasmic face with nuclear envelope stripped off.
Scanning electron micrographs of the nuclear pore complex
Cytoplasmic face
note the octagonal arrangement
Nucleoplasmic face
displays a basket like structure in side the nucleus
Nucleoplasmic face with nuclear envelope stripped off.
connections to the nuclear lamina

28. Reconstruction of nuclear pore complex
Alberts 12-10
Reconstruction of nuclear pore complex
Column component forms the walls of the pore
Annular subunit protrudes into the center of the pore
Luminal subunit formed by a transmembrane glycoprotein that anchors pore in the membrane
The ring subunit interacts with nuclear lamina also anchoring it in place.
Fibrils extend from both the cytoplasmic face and the nucleoplasmic face (basket or cage like structure)
Reconstruction of nuclear pore complex
Column subunit
Annular subunit
Luminal subunit

29. How big is the pore Alberts 12-12
Experimental results indicate diameter is 9 nm
EM data my be missing a key component
Central transporter or plug
How big is the pore?
To evaluate this, labeled molecules were injected into cells and the rate of entry into the nucleus was measured. Rate depends on the size of the molecule. Determined that most proteins are too large to pass through the pore.
Experimental results indicate diameter is 9 nm
EM data my be missing a key component
Central transporter or plug

30. So how do proteins get through the pore?
NLS
Nuclear localization signal
NES
Nuclear exclusion signal
NRS
Nuclear retention signal
NLS--determined by experimental observations
Nuclear localization signal
NES--leucine rich sequences
Nuclear exclusion signal
No clear consensus
NRS--Binding site for nuclear substructure
Nuclear retention signal

31. Nuclear localization signal
Alberts 12-13
Nuclear localization signal
SV40 T-antigen normally imported into nucleus
Single mutation inhibits transport
Smith 1984

32. Linear signals Bipartite signals Cooper 8-8
Nuclear localization signal can be a linear string of amino acids or can be separated along the primary structure (bipartite signal). The bipartite signal “parts” are closely arranged in the three dimensional structure of the protein.
Linear signals
Bipartite signals

33. How do we know this is actually do to transport through the pores?
Experimentally!
Inject radiolabeled protein and determine what gets into nucleus
Gold label nuclear targeted protein and examine by EM
How do we know this is actually do to transport through the pores?
Experimentally!
Inject radiolabeled protein and determine what gets into nucleus.
use autoradiography to visualize cell
Gold label nuclear targeted protein and examine by EM
Alberts 12-14

34. Gold label associated with nuclear pores along the nuclear envelope
Experimental result with gold labeled protein. Can see a great deal of gold label associated with the nuclear envelope.
Alberts 12-15

35. Exposing NLS results in transport
Alberts 12-19
NLS can be masked
Hormone receptors found in cytosol until activated by hormone.
Exposing NLS results in transport
NLS can be masked
Hormone receptors found in cytosol until activated by hormone.
Glucocorticoid receptor is associated with a chaperonin protein.
Binding of hormone to receptor releases chaperonin and exposes NLS
Exposing NLS results in transport and binding of the receptor to DNA
Net result is the activation of transcription.

36. What is happening at the molecular level?
Step 1: Importins
Import receptors a and b
Importin a bind to NLS
Importin-cargo complex binds to the cytoplasmic fibrils of the nuclear pores
Does not expend energy

37. What is happening at the molecular level?
Step 2: Ran GTPase cycle
Ran relative of Ras is a small GTPase
Activity depends on whether GDP or GTP is bound
Ran GTP in the nucleus
Binds importins inside the nucleus
Releases importins from cargo
Ran GDP in the cytoplasm

38. RanGTP binds importins RCC1: Ran guanine exchange factor
Active form
RanGAP
RanGTP binds importins
Hydrolysis of GTP results in the release of importins from Ran
RCC1: Ran guanine exchange factor
Replacement of GDP by GTP
RanGAP: Ran GTPase activating protein
Activates GTP hydrolysis
RanGTP binds importins
Hydrolysis of GTP results in the release of importins from Ran
RCC1: Ran guanine exchange factor
Replacement of GDP by GTP
RanGAP: Ran GTPase activating protein
Activates GTP hydrolysis

39. Import Lodish 11-37 Importins bind cargo with NLS
Associate with the fibrils of pore complex
High concentration of RanGTP in nucleus stimulates transport through pore
RanGTP binds importins inside nucleus
Release of cargo
Recycle importins and Ran
Import
Importins bind cargo with NLS
Associate with the fibrils of pore complex
High concentration of RanGTP in nucleus stimulates transport through pore
RanGTP binds importins inside nucleus
Release of cargo
Recycle importins and Ran
Importin a can freely pass through the pore
Importin b-RanGTP complex pass through the pore
Hydrolysis of GTP releases importin b into the cytoplasm
Lodish 11-37

40. Export Exportin binds NES of cargo RanGTP binds exportin
Stimulates transport through the pore
GTP hydrolysis releases cargo in the cytoplasm
Ran and exportin are recycled
Export
Exportin binds NES of cargo
Exportins are related to importin b
RanGTP binds exportin
Increases affinity of exportin for cargo
Stimulates transport through the pore
GTP hydrolysis releases cargo in the cytoplasm
Ran and exportin are recycled
Both can pass freely through the pore
Lodish 11-33

41. Lodish 11-34
RNA export
mRNA not transported until after splicing, polyadenylation and capping
In the nucleus, mRNAs are coated with hnRNPs
Some hnRNps contain NRS
Others are exported with message and transported back into the nucleus
Protect the flow of genetic information
RNA export
mRNA not transported until after splicing, polyadenylation and capping
In the nucleus, mRNAs are coated with hnRNPs
Some hnRNps contain NRS
Others are exported with message and transported back into the nucleus
Protect the flow of genetic information